The long-term objectives of the proposed work are to elucidate the mechanisms by which hyperglycemia-induced intracellular reactive oxygen species (ROS) produce diabetic retinopathy. The specific research proposed in this application will elucidate the role played by ROS-induced glyoxalase I substrates, which are precursors of intracellularadvanced glycation endproducts and mediators of hyperglycemia-induced angiopoietin-2 gene expression in retinal Muller cells. Specific Aim 1 will evaluate the effect of hyperglycemia-induced intracellular reactive oxygen species (ROS)on glyoxalase I substrates, advanced glycation endproducts derived from these substrates, and diabetic retinopathy in uncoupling protein-2 (UCP-2) knockout and glyoxalase I transgenic mice. Streptozotocin diabetes will be induced in UCP-2 knockout, glyoxalase I transgenic, and wild type mice. Retinal concentrations of intracellular oxidative stress products, ROS-induced glyoxalaseI substrates, and glyoxalase-I substrate-derived AGEs will be determined in diabetic and non-diabetic mice. Retinopathy will be assessed by semi-quantitative RT-PCR and in situ hybridization at early time-points, and by quantitativemorphology at late time-points. Specific Aim 2 will evaluate the effect of inhibition of hyperglycemia-induced ROS on glyoxalase I substrates, advanced glycation endproducts derived from these substrates, and diabetic retinopathy in uncoupling protein-2 (UCP-2) knockout, glyoxalase I transgenic, and wild type mice. Streptozotocin diabetes will be induced in UCP-2 knockout, glyoxalase I transgenic, and wild type mice. In selected groups of these animals, hyperglycemia-induced intracellular ROS will be inhibited by treatment with either MnTBAP or EUK8, structurally distinct SOD/catalase mimetic compounds. Retinal endpoints will be assessed as described in Specific Aim 1. Specific Aim 3 will characterize the mechanism of Angiopoietin-2 transcriptional control by glyoxalase I substrates in cultured primary retinal Muller cells. A hyperglycemia-responsive mouse angiopoietin-2-luciferase reporter vector has been constructed which contains 2.5kb 5'-flanking sequence. Progressive deletions from the 5' end of the Ang-2 promoter will be constructed using restriction endonucleases or PCR methods. The glyoxalase-I substrate responsive region will be identified, and known transcription factor binding sequences altered by site-directed mutagenesis. EMSA will be performed with indicated consensus oligonucleotides from the glyoxalase-I responsive region. Supershift experiments will use commercially availableantibodies. IP-westernsof cell extracts will be immunoblotted with antibodies to glyoxalase I-substrate-derived advanced glycationendproducts.